Role of p-lactamase Mutations in Antibiotic Resistance Bacterial resistance to antimicrobial agents has increased in recent years and now represents a significant threat to successful antibiotic therapy. One example of this phenomenon is the development of resistance to p-lactam antibiotics, p-lactam antibiotics, such as the penicillins and cephalosporins, are among the most frequently used antimicrobialagents. The most common mechanism of resistance to P-lactam antibiotics is the production of p-lactamases, which cleave the antibiotic, rendering it harmless to bacteria. Based on primary sequence homology, p-lactamases have been grouped into four classes. Classes A, C and D arc active-siteserinc enzymes that catalyze, via a serine-bound acyl-enzyme intermediate,the hydrolysis of the p-lactam antibiotic. Class B enzymes require zinc for activity and catalysis does not proceed via a covalent intermediate.Because of the diverse range of substrate specificities of these enzymes, virtually all p-lactam antibioticsaresusceptible to hydrolysis. Clearly, the design of new antibiotics that escape hydrolysis by the growing collectionof \\- lactamase activitieswill be a challenge. It will be necessary to understand the catalytic mechanism and basis for substrate specificity of each class of p-lactamase. The goal of this work is understand how the amino acid sequence determines the structure, catalysis, and substrate specificityof the IMP-1 p-lactamase of class B and the P99 class C p-lactamasc. This will be achieved by randomizingamino acid positions in theactive-site pocket of each en/yme to sample all possible aminoacid substitutions. All of the random substitutions will then be screened to identify those substitutionsthat alter the substrate specificity of the enzyme. Enzymes containing substitutions that alter substrate specificitywill be purified and characterized biochemically. The sets of random substitutionswill also be screened using phage display methodology to identify residues critical for catalysis. A further goal of this proposal is to use the detailed knowledge of the interface between p- lactamase inhibitoryprotein (BLIP) and p-lactamasc, in combinationwith random mutagenesis and phage display, to create derivatives of BLIP that bind and inhibit P-lactamases and penicillin binding proteins. The new BLIP derivatives will be characterized biochemically and structurally. The informationgained from these studies will be useful for the rational design of new antibioticsand inhibitors.